In the United States reported human vector-borne diseases are primarily tick-borne. For continued cycling of tick-borne disease pathogens in nature, successful tick feeding is required. Molecular mechanisms that regulate early stage tick feeding are poorly defined. They are critical to understanding the acquisition and transmission of pathogens by ticks, which in turn will be important in designing novel approaches to control ticks and tick- borne diseases. The goal of this proposal is molecular identification and target validation of Amblyomma americanum tick saliva proteins that are injected into the host during the first 48 h of tick feeding. The rationale to focus on this time point is that, it precedes the key facets of tick feeding, blood meal up take and tick borne disease agent transmission. The hypothesis is that tick saliva proteins critical to feeding success and pathogen infection of the host are injected into the host during the first 48 h of feeding and blocking their functions will protect animals against tick feeding and pathogen infection. This hypothesis is based on preliminary experimental evidence that repeated tick infestation of rabbits for 48 h with nymph or adult ticks conferred protective tick immunity as revealed by mortality failure of ticks to attach or remain attached onto host skin. Previous efforts to confer protective tick immunity by immunizing animals with single tick saliva recombinant proteins have produced mixed results. In this research we have proposed a previously unexplored approach, to immunize animals against tick feeding with multi-epitope chimeric tick saliva protein vaccine antigens. The idea is that immunity to these chimeric antigens will mimic protective tick immunity conferred tick saliva protein antigens during repeated infestations. There are 3 specific aims. The first is to clone cDNAs that encode A. americanum tick saliva proteins that are injected into the host during the first 48 h of tick feeding. The second is to validate immunogenicity of putative antigenic peptide regions in tick saliva proteins. Validated immunogenic peptides will represent tick saliva protein regions that mediate tick resistance in repeated tick feeding and will thus represent potential tick vaccine antigens. The third is to validate tick immunity and anamnestic antibody response of immunogenic peptide-cocktail immunized rabbits to tick infestation. The rationale is that if immunization of rabbits with immunogenic peptide-cocktails confers tick immunity that is comparable to that in repeated tick infestation, data from this application will set the foundation to design chimeric tick saliva proteins.